Capillary electrophoresis has proved to be a highly effective means of analyzing extremely small biological samples. The long separation path in a capillary permits the separation of a multitude of components in a single sample, including components which are closely related. In addition, the thin diameter of a capillary permits the use of high voltages, which produce separations in a relatively short period of time. Furthermore, capillaries are particularly well suited for on-line detection of the separated species by passing a light beam through the capillary itself directly into a detector.
The unusually high resolution obtained in capillary electrophoresis is attributable to the small internal radius of the capillary which facilitates the efficient removal of the joule heat generated by the high-voltage electric current. As the separations attempted in capillaries become increasingly sensitive, the need for efficient heat transfer from the capillary becomes more significant. In coolant systems in general, liquid cooling is far more efficient than air cooling, and it is reasonable to expect that this would apply to capillaries as well.
Capillary cartridges designed for circulating liquid coolant have indeed been developed, but they suffer from a number of disadvantages. Due to the geometries involved, it is far more difficult to devise a liquid-cooled capillary cartridge than an air-cooled cartridge. The reasons include the fact that the ends of the capillary must be electrically isolated so that electrical potential differences of up to 30,000 V can be applied. Furthermore, the tips of the capillaries must extend into reservoirs containing buffer solutions which serve as liquid junctions to communicate the capillary tips with electrodes. Still further, to be adaptable for use with a variety of sample mixtures of different compositions, the capillary must be exchangeable with capillaries of different lengths and diameters. Capillary lengths in common use, for example, range from 20 cm to 100 cm.
In its most widely used configurations, the capillary is arranged with its tips directed vertically downward and terminating at the same elevation, for purposes of hydrostasis. The capillary in these configurations thus forms an arch terminating at the downward-directed tips. In water-cooled systems presently known, the capillary is coiled within a cartridge housing of relatively compact volume through which the liquid coolant circulates. Coiling permits a single cartridge housing to accommodate different capillary lengths, while facilitating efficient cooling and reducing the overall dimensions of the cartridge. This however limits the selection of different capillary lengths to multiples of the coiling circumference, and the circumference itself is generally dictated by the size and structure of the cartridge. Furthermore, the coiling prevents the achievement of uniform heat transfer, despite the construction of coolant flow conduits in the capillary which are shaped to direct and confine the coolant to the vicinity of the capillary coils.
A further problem with the coiling of capillaries is what might be termed the "race-track" effect, i.e., the difference in path length along the capillary as a function of cross-sectional position, ranging from a relatively short path closest to the center of the coil to a relatively long path furthest from the center. The degree of difference and hence the severity of the effect are proportional to the number of turns in the coil. This can be significant, since coils of several turns are commonly used to achieve optimal resolution and separation of the solute peaks. Recent studies have confirmed that coiled capillaries suffer a loss of resolution relative to straight capillaries run under otherwise identical conditions. As more sensitive separations are attempted and performance expectations increase, the race-track effect will take on greater significance and its avcoidance will become more important.
The need to immerse the capillary tips in the buffer reservoirs raises a further consideration in liquid-cooled systems. To permit the immersion, the tips protrude from the cooled volume through ports which are sealed around the outer diameter of the capillary with wax or glue. Any areas which are left uncooled introduce errors and variations in the detection results. Since the protruding tips are uncooled, cartridges of this type must be designed so that the ratio of the combined lengths of the protruding tips to the total capillary length is as small as possible.
Another major consideration in capillary electrophoresis is the accurate alignment of an optical detection system with the capillary wall for on-line detection. At least one window along the capillary must be provided for optical detection, and it must be properly aligned with the lenses in the cartridge housing which form the light path through the cartridge. In systems designed for capillary exchangeability, the ease of alignment is even more critical since proper alignment must be achieved each time the capillary is changed. In liquid-cooled cartridges currently known, the segment of the capillary which is designated as the window is aligned in a precise manner with a fixed external window in the cartridge envelope or housing, then sealed with wax or adhesive to isolate the external surface of the segment from the coolant. Accurate alignment assures reproducible results but requires significant time and detailed effort.
What does not presently exist, and is strongly needed, is the means to rapidly and conveniently exchange capillaries in a cartridge while maintaining efficient cooling without coiling or tedious optical alignment procedures. A system which can meet these requirements will significantly help capillary electrophoresis to achieve its full potential.